mRNA molecules are composed of four main elements: a 5' cap, a 5' untranslated region (5' UTR), an open reading frame (ORF), and a 3' untranslated region (3' UTR) followed by a poly(A) tail. The 5' cap is a modified nucleotide that protects the mRNA from degradation by exonucleases and facilitates its recognition by the translation machinery. The 5' UTR is a non-coding sequence that regulates the translation initiation and efficiency of the mRNA. The ORF is the coding sequence that encodes the protein of interest. The 3' UTR is another non-coding sequence that influences the stability, localization, and translation of the mRNA. The poly(A) tail is a stretch of adenine nucleotides that enhances the stability and export of the mRNA from the nucleus. These elements are essential for the function and fate of mRNA in the cell and, therefore, need to be carefully designed and optimized for biomedical applications.
Fig.1 Structural features of IVT mRNA. (Rein Verbeke, 2019)
One of the most common and effective ways to modify mRNA constructs is to alter the 5' cap structure. The 5' cap is a modified nucleotide that protects the mRNA from degradation by exonucleases and facilitates its recognition by the translation machinery. The natural 5' cap of mRNA is a 7-methylguanosine (m7G) linked to the first nucleotide of the mRNA by a 5'-5' triphosphate bridge. However, this type of cap is not optimal for in vitro transcribed (IVT) mRNA, as it is prone to hydrolysis and degradation. Therefore, various synthetic 5' cap analogs have been developed and used to improve the stability and translation efficiency of IVT-mRNA. For example, anti-reverse cap analogs (ARCAs) are modified nucleotides that have a 3'-O-methyl group that prevents the formation of the reverse orientation of the cap, which is detrimental for translation. Another example is the coronavirus (CoV) RNA cap, which is a 2'-O-methylated nucleotide that mimics the cap structure of CoV RNAs, which are known to evade the innate immune system. Both ARCAs and CoV RNA caps have been shown to enhance the stability, translation efficiency, and reduced immunogenicity of IVT-mRNA in various applications.
Another important aspect of mRNA construct optimization is the design and modification of the 5' and 3' untranslated regions (UTRs). The 5' and 3' UTRs are non-coding sequences that regulate the translation initiation and efficiency of the mRNA. The 5' and 3' UTRs can be derived from natural sources, such as viral or cellular mRNAs, or from synthetic sources, such as computer-generated sequences. The choice of the 5' and 3' UTRs depends on the desired properties and functions of the mRNA. For example, some viral 5' and 3' UTRs, such as those from the encephalomyocarditis virus (EMCV) or the hepatitis C virus (HCV), can enhance the translation of the mRNA by forming internal ribosome entry sites (IRES) that bypass the need for the 5' cap recognition. However, these viral UTRs may also induce unwanted immune responses or interfere with the host gene expression. Therefore, synthetic 5' and 3' UTRs have been designed and tested to achieve optimal translation efficiency and reduced immunogenicity of the mRNA. Moreover, the 5' and 3' UTRs can also incorporate various regulatory elements, such as microRNA (miRNA) binding sites, RNA stability elements, or RNA localization signals, that can modulate the expression and function of the mRNA in a spatial and temporal manner.
1. The impact of different 5' cap modifications on mRNA stability, translation efficiency, and immunogenicity
The 5' cap modification is a crucial factor that affects the stability, translation efficiency, and immunogenicity of mRNA constructs. Different 5' cap modifications have different advantages and disadvantages in terms of these aspects. The conventional m7G cap is the most widely used and well-studied cap analog, but it also has some drawbacks, such as triggering innate immune responses by activating TLR7 and TLR8, or inducing type I interferons. Alternative 5' cap modifications, such as ARCA, CoV-RNA, or m1Ψ, have been developed to overcome these limitations and improve the performance and safety of mRNA constructs.
The ARCA cap is a modified nucleotide that has a 3'-O-methyl group that prevents the formation of the reverse cap structure, which can impair the translation efficiency of the mRNA. The ARCA cap has been shown to improve the stability and translation efficiency of mRNA constructs compared to the m7G cap, especially in the presence of cap-binding proteins such as eIF4E or eIF4G. However, the ARCA cap still has some immunogenicity issues, as it can activate TLR7 and TLR8 or induce type I interferons, albeit to a lesser extent than the m7G cap.
The CoV-RNA cap is a synthetic cap analog that mimics the structure of the 5' end of coronavirus RNA, which has a 1'-2'-O-methylated ribose and a 2'-O-methylated cytidine. The CoV-RNA cap has been demonstrated to improve the stability, translation efficiency, and immunogenicity of mRNA constructs compared to the m7G cap. The CoV-RNA cap can protect the mRNA from degradation by exonucleases, enhance its binding to the translation initiation complex, and reduce its recognition by TLR7 and TLR8. Moreover, the CoV-RNA cap can also increase the expression of the encoded protein in various cell types and animal models.
The m1Ψ cap is a naturally occurring cap analog that has a methyl group at the N1 position of pseudouridine. The m1Ψ cap has been shown to enhance the stability, translation efficiency, and immunogenicity of mRNA constructs compared to the m7G cap. The m1Ψ cap can increase the stability of the mRNA by forming more stable secondary structures, improve the translation efficiency of the mRNA by facilitating its interaction with the translation initiation complex, and reduce the immunogenicity of the mRNA by inhibiting the activation of TLR7 and TLR8. Furthermore, the m1Ψ cap can be combined with other nucleoside modifications, such as pseudouridine or 1-methylpseudouridine, to further improve the performance and safety of mRNA constructs.
2. The influence of different 5' and 3' UTR sequences or elements on mRNA localization, translation initiation, and degradation
The 5' and 3' UTR sequences or elements are another important factor that affects the localization, translation initiation, and degradation of mRNA constructs. Different 5' and 3' UTR sequences or elements have different effects on these aspects. The 5' and 3' UTR sequences or elements can be derived from natural or synthetic sources, or can be designed to incorporate specific regulatory features. The optimization and modification of the 5' and 3' UTR sequences or elements can have a significant impact on the performance and safety of mRNA constructs.
The 5' UTR sequences or elements can influence the localization and translation initiation of the mRNA by affecting its secondary structure, stability, and interaction with various factors, such as ribosomes, initiation factors, or microRNAs. The 5' UTR sequences or elements can be optimized or modified to enhance the translation efficiency or specificity of the mRNA. For example, the 5' UTR of the EMCV or the HCV contains an IRES that can mediate the cap-independent translation initiation of the mRNA, which can increase the translation efficiency of the mRNA in the presence of cellular stress or viral infection. The 5' UTR can also contain uORFs or leader peptides that can regulate the translation initiation of the downstream ORF by affecting the scanning or reinitiation of the ribosome, which can modulate the translation specificity of the mRNA depending on the cellular context or stimuli.
The 3' UTR sequences or elements can influence the translation termination, stability, and localization of the mRNA by affecting its secondary structure, degradation, and interaction with various factors, such as poly(A) binding proteins, deadenylases, microRNAs, or RNA-binding proteins. The 3' UTR sequences or elements can be optimized or modified to protect or target the mRNA for degradation or localization. For example, the 3' UTR of the WHV or the BGH contains stabilizing elements that can protect the mRNA from degradation by deadenylases or exonucleases, which can increase the stability and expression of the mRNA. The 3' UTR can also contain AREs or MREs that can regulate the translation or degradation of the mRNA by binding to RNA-binding proteins or microRNAs, which can fine-tune the expression level or duration of the mRNA.
One of the major challenges of mRNA construct design is to balance the stability and immunogenicity of the mRNA. Stability refers to the ability of the mRNA to resist degradation and maintain its function in the cell. Immunogenicity refers to the ability of the mRNA to elicit an immune response from the host. Both stability and immunogenicity are influenced by various factors, such as the 5' cap, the 5' and 3' UTRs, the ORF, the poly(A) tail, and the nucleoside modifications of the mRNA. However, there is often a trade-off between these two properties, as some modifications that enhance the stability of the mRNA may also increase its immunogenicity, and vice versa. For example, the CoV RNA cap, which is a 2'-O-methylated nucleotide that mimics the cap structure of CoV RNAs, can improve the stability and translation efficiency of the mRNA, but it can also trigger an innate immune response by activating the pattern recognition receptors (PRRs) that recognize CoV RNAs. Similarly, the poly(U) tail, which is a stretch of uridine nucleotides that replaces the poly(A) tail, can increase the stability and export of the mRNA, but it can also induce an inflammatory response by activating the toll-like receptor 7 (TLR7) that recognizes single-stranded RNA. Therefore, it is important to carefully select and combine the appropriate modifications for the mRNA construct, depending on the intended application and the desired outcome.
Another key aspect of mRNA construct optimization is to enhance the targeting and delivery efficiency of the mRNA. Targeting refers to the ability of the mRNA to reach the specific cell type or tissue of interest. Delivery refers to the ability of the mRNA to enter the cell and escape the endosomal compartment. Both targeting and delivery are crucial for the efficacy and safety of the mRNA, as they determine the expression and function of the encoded protein in the cell. However, both targeting and delivery are challenging to achieve, as the mRNA is a large and negatively charged molecule that faces many barriers and obstacles in the biological environment. Therefore, various strategies and methods have been developed and used to improve the targeting and delivery of the mRNA. For example, the mRNA can be conjugated or complexed with various molecules or materials, such as peptides, antibodies, lipids, polymers, or nanoparticles, that can facilitate the binding, uptake, and release of the mRNA in the target cell. Another example is the use of viral vectors, such as adenovirus, lentivirus, or adeno-associated virus, that can deliver the mRNA into the cell by exploiting the natural infection mechanism of the virus. Both conjugation and complexation methods have been shown to enhance the targeting and delivery efficiency of mRNA in various applications.
Messenger RNA (mRNA) is a promising platform for gene delivery, protein expression, and vaccine development, as it can induce potent and specific immune responses as well as produce functional and therapeutic proteins. However, mRNA also faces several challenges and limitations, such as instability, low translation efficiency, and immunogenicity, that hamper its clinical applications. Therefore, the design and development of mRNA constructs is a critical and active area of research, aiming to improve the performance and safety of mRNA-based therapies.
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